Ординатура / Офтальмология / Английские материалы / The Glaucomas Volume 1 Pediatric Glaucomas_Sampaolesi, Zarate_2009

We should keep in mind that this classification is important for the pathologist and for an accurate clinical and pathological correlation, and not for the filtering results, which in general are similar (Fig. 26.1).

Before analyzing the general and particular criteria of each structure mentioned and to complete the list of surgical material that enables the pathological study of the anterior segment in glaucoma, we must mention irido-cyclo-trabeculectomy, an operation described by Jorge Malbran, with more than 40 cases studied in the same period as the 1,800 trabeculectomies. Iridotrabeculectomy is actually a goniectomy, as the posterior cut of the specimen passes through the anterior ciliary furrow, and so the iris root is included within the

specimen. This surgery, as we shall see later, is preferably indicated in neovascular glaucomas.

Recently, the so-called nonpenetrating deep sclerectomy has been added to the study of these tissues, where the two important structures to identify are (1) the second scleral flap, which, when well performed, should include the endothelial surface of the outer wall of the Schlemm canal, which we can easily identify with hematoxylin and eosin, and (2) in difficult cases CD34 or factor VIII, which are the endothelial markers studied by immunohistochemistry.

After this general panorama of the surgical and pathological technique, a detailed description of the technical methodology and the findings will be provided.

Requirements for Sending

Glaucoma Pathology Samples

1.Biopsies. The samples obtained from biopsies (trabeculectomies, nonperforating deep sclerectomy, and irido-cyclo-trabeculectomies) must be fixed in 10% formaldehyde (except in cases where transmission electron microscopy is decided on from the start. In these cases, the fixative is glutaraldehyde, with its modifications). The quantity and quality of the fixative should be checked periodically. These specimens should be placed in small, transparent flasks so that they are permanently visible. The specimens are measured along their three axes: anteroposterior and transversal. The thickness is very difficult to measure and is done in the histological preparation. The specimens are photographed with a surface optical microscope, following a technique we have described, which will be explained below.

2.Surgical specimens. Enucleation or autopsy eye. just as in the biopsies, these are fixed in 10% formaldehyde, paying close attention to the volume of the container (glass or plastic bags). These should let the fixative liquid be approximately 20 times the volume of the specimen.

3.Anterior chamber cytologies. The best technique, which we always use in our laboratory, is to place any cytological material in preserving liquid, and then use the robot cytocentrifuge to perform the monolayer extended liquid technique, which enables the greatest cell concentration possible. To identify the materials, we recommend writing on the labels in pencil, since this prevents any liquid leakage. All material sent should be accompanied by the corresponding request for an examination with as much data as possible of all kinds, and photocopies or imaging studies made previously. These data are extremely important in making the best and most detailed histopathology report possible.

Macroscopic Study of the Specimens

As mentioned above, in all cases the macroscopic study of the specimens was made with surface optic microscopy, in a technique that we presented in 1990 in the 18th Congress of the International Academy of Pathology. This is based on two fundamental principles performed in the material inclusion process:

1.Transparency of the specimen when it is passed through Xylol;

2.Application of the Scheimpflug principle, which enables good-resolution documentation to be obtained of the different planes with great depth of focus.

Methodology of Trabeculectomy Structure Study

In all cases, a drawing of the specimen is made, which, together with the surface optical microscopy photo, enables the inclusion to be performed in the most suitable way, i.e., with the transversal axis of the specimen perpendicular to the histological cross-section, ensuring the classic anatomy of the chamber angle. Between 20 and 100 semiserial sections are made, depending on the size and interest of the material.

At least eight semiserial sections are placed in a holder, which sometimes makes it possible to detect the sequence, for example of a pigmentary thrombotic process.

Special techniques are used with the remaining sec- -tions:

Masson’s trichrome. This is useful because it marks out the fibroconnective tissues in blue and the muscle in red, which, in the case of an anterior surgical limit trabeculectomy, can be so scant that the hema-

-toxylin-eosin does not show it.

Gomori’s reticulum. This technique is very useful to show the reticular fibers and procollagenase, present

-in the pretrabecular area of congenital glaucomas. PAS (periodic acid-Schiff). PAS stains the neutral mucopolysaccharides. This marker is useful to show

-the basal membranes.

Alcian blue. This technique stains the acid mucopolysaccharides. Associated with trichrome and PAS, it defines certain characteristics in congenital glaucoma.

In the past 10 years, we have been using immunohistochemical techniques for cases requiring it, to make the structural alterations more evident. An example is the use of CD34 to mark small vessels that can pass unnoticed with hematoxylin and eosin and the use of desmin for muscular fibers, etc.

In some cases, we have performed transmission electron microscopy in material already included in paraffin. Even though the documentation is not the best, it can be useful in certain types of diagnoses.

Study of the Irido-cyclo-trabeculectomy

As with the trabeculectomies, surface optical microscopy photographs, a small prior sketch following the paraffin inclusion process are the basis for histological preparations with the smallest number of artifacts possible. In these specimens, great attention should be paid to the chamber angle and, as already specified, to detecting the vascular or fibrous membranes that may be found in this area with the use of conventional

or special techniques. The data that can be provided by the iris root naturally adhering to the dried surgical specimen are very interesting. For the pathologist, this is, in this sense, the most significant specimen for panoramic and minute observation of all the structures related to the aqueous humor drainage system.

Study of Nonperforating Deep Sclerectomy

As regards the study of nonperforating deep sclerectomy (NPDS), we will go further into this in the study of nonpediatric glaucoma, but we will give a few useful rules related to what the pathologist is looking for in these specimens.

The biopsies are fixed in 10% formaldehyde. Then the biopsies are dehydrated in three consecutive 2-h steps, each in 96% alcohol and one 1-h step in 100% alcohol. They are then passed through xylol for 2–3 h. The material at this stage is placed in a holder under a magnifying glass to identify the corresponding zones, marking the endothelial structures, for their subsequent orientation in paraffin. The photographs are taken using the method described. The specimen is cut by freezing to confirm the position of the endothelium and is included. The third sample sent, consisting of the internal wall of the Schlemm canal and the outer half of the corneoscleral trabecular meshwork is very difficult to handle technically, because of its friability and size. We will devote a deeper study to this in nonpediatric glaucomas (Volume II: Primary Glaucomas, in preparation).

The following histological sections correspond to congenital, especially refractory, glaucomas, which were stained with the techniques described above.

Microscopy of Trabeculectomy Specimens

Surface Light and Electron Microscopy

and Scanning Microscopy

When either goniotomy or trabeculectomy are the techniques used, no material can be obtained for pathological examination. Specimens for this purpose can only be obtained in trabeculectomies. It should be kept in mind that we use combined procedures (trabeculotomy + trabeculectomy) or only trabeculectomy in cases in which the axial length has already grown considerably, over 24 mm within the first 6 months of life, generally with corneas 13 mm or larger and endothelium and Descemet membrane tears. This means that anatomopathological evaluations can only be made in advanced cases.

Anatomopathologic results were presented for the first time in 1977 [1] and 1979 [2]. Numerous schools have also analyzed trabeculectomy specimens subsequently.

Material Collection

To obtain adequate specimens for analysis, we believe the surgical technique used should be very thorough and careful. During surgery, it is vital to use Minsky’s transillumination for proper placement of the incision, opening the anterior chamber exactly at the corneal endothelium immediately beyond the Schwalbe line.

Using the Minsky technique, the cornea in the illuminated area can be 1 mm away from the light-dark- ness limit.

After careful dissection of a limbal-based scleral flap with a thickness half that of the corneoscleral thickness, two lateral incisions are marked perpendicular to the limbus. After opening the anterior chamber (third incision), these perpendicular incisions are completed with angled Vannas scissors. The trabeculectomy specimen is secured with a fine-toothed clamp for microsurgery, at half the thickness facing the external surface, in order to avoid damage to the fine trabecular structure. This is an open procedure and the surgeon can see the external wall of the chamber angle up to the spur. The final incision is made with the same Vannas scissors between the scleral spur and the ciliary body band. From the corneal incision to the last incision, the assistant should carefully and gently put saline drops on the specimen.

The specimen for light microscopy is kept in a bottle with 10% formol solution. After fixing it, it is embedded in paraffin and stained according to the following techniques: (1) hematoxylin-eosin, (2) Masson, (3) PAS, and (4) Gomori’s and Del Rio Ortega’s technique for reticulin determination. In each specimen, from 120 to 300 sections are made depending on size. The first 30 sections are stained with hematoxylin-eosin to determine whether the orientation of the sample is correct. Should this be the case, they are stained on three slides of 30 sections each with special techniques. If these stainings are incorrect, the rest are stained with hematoxylin. We have recently adopted fixation in 96°–100° alcohol, with the consequent addition of immunomarking techniques to the previous staining methods.

The specimen for electron microscopy is fixed immediately in the operating room, as explained above, by immersing it into 25% glutaraldehyde solution and a buffer solution (Millong’s phosphate 0.1 M, with glucose, pH 7.4, for 24 h). Then it is washed with buffer

isotonic solution for 15 min and fixed again in a solution of osmium peroxide 1% in a Millong’s phosphate buffer with glucose, pH 7.4, for 2 h. After this, the specimen is dehydrated in acetone solutions of different degrees and dried according to the critical point method in CO2, with the Sorvall system. Using this method, acetone is replaced by carbon dioxide in a high-pressure chamber. Once dried, the trabeculectomy specimen is placed on a device supporting it with the trabecular surface upward and it is covered with a fine coat of carbon and gold palladium, using a Geol vacuum unit. These specimens were examined with a scanning electron microscope, model T.S.M.-U.

Material Examined

The material examined is described in Table 26.1, where, in addition to age, gender, and intraocular pressure, echometric and corneal diameter values are reported, as well as whether there are endothelium or Descemet membrane tears reflecting the degree of disease progression.

In addition, four specimens obtained from young 15-year-old patients and one from a 32-year-old patient with late congenital glaucoma, have been studied.

Table 26.1 lists the most important clinical data of the first specimens studied. The clinical–pathologic correlation is the only way to advance the knowledge of the disease.

Table 26.2 lists the anatomopathologic findings in the first 13 cases. Furthermore, another 53 specimens were studied, but they were not included in the table for the sake of brevity.

Terminology

As already mentioned, in gonioscopy as well as in pathologic anatomy, there are many different terms to refer to the anomalous tissue located in the chamber

-angle. These terms were:

Pectinate ligament. Given the similarity found by some authors with this anatomic formation exclusively belonging to ungulates (horses), at present we

-call them pathological mesodermal remnants.

The Barkan membrane. This term is used to name a membrane discovered by Barkan, which is seen through the gonioscope and evidenced during surgery, because it is difficult to histologically demonstrate its presence. With electron microscopy, Jerndal demonstrated that it was a membrane covering the anomalous tissue formed by a layer of polygonal

-cells.

Anterior insertion of the iris, high insertion of the iris. This term was coined by numerous authors, though it is an apparent image, since the iris root, only formed by its deep mesenchymal layer, always inserts in the same place: the ciliary body band,

formed by the inner surface of this muscle. However, the anomalous tissue attaches to the actual insertion spot and always to the same place in the iris, and may reach as far as the spur; sometimes it extends to the trabecular meshwork and other structures. It can even cover the Schwalbe line by surpassing it, thus producing the appearance of an anterior or

-high insertion.

Pathological mesodermal remnants. This is the terminology we currently prefer, as do other authors, since it is a mesodermal tissue remaining in the chamber angle that has failed to be resorbed before month 9 of gestation, in which reticulin fibers prevail, like the tissue which is normally located there in fetal life. In contrast, in normal mesodermal remnants, known as iridian processes, collagen fibers prevail and may be present in any subject.

The term used to name this tissue in the first edition of this book has thus been changed, since at that time the term “pectinate ligament” was used, following Busacca’s concept.

Normal Mesodermal Remnants:

Iridian Processes

Iridian processes are formed by a central collagen axis and a very small number of reticulin fibers with fibroblasts surrounded by numerous melanocytes. In other words, their structure is identical to that of the superficial mesodermal layer of the iris (Fig. 26.2).

The iridian processes extend from the inner wall

of the chamber angle (iris) to the external wall (sclera and cornea). The iridian edge of these processes never reaches beyond the base of the last circular fold of the iris, i.e., they never originate in the iris root. The corneoscleral end may reach different heights of the external wall of the chamber angle: the corneoscleral trabecular meshwork, the scleral spur, the trabecular meshwork at the Schlemm canal or the Schwalbe line, but they never reach as far as the latter.

Pathologic Mesodermal Remnants

Pathologic mesodermal remnants are found in pure congenital glaucoma, refractory congenital glaucoma, and late congenital glaucoma (goniodysgenesis) at 6–40 years of age.

As a primary concept, it should be stressed that there are pronounced differences in the pathologic anatomy

-of three types of glaucomas.

In pure congenital glaucomas, these remnants are diffuse, in chamber angle type I and apparent high

-insertion of the iris in type II.

In late congenital glaucomas, there are mesodermal pathological remnants from the scleral spur to the iris root. The ciliary band is not visible.

Diffuse Mesodermal Remnants:

Pure Congenital Glaucoma

Diffuse mesodermal remnants in pure congenital glaucoma (70% of cases) have frequently been found in the chamber angle of pure congenital glaucoma specimens (Fig. 26.3).

In gonioscopy, they have the appearance of a tissue band that is seen around the entire circumference of the chamber angle, covering the structures of the external wall to different extents, from the roots of the iris, ciliary body band, the scleral spur, and sometimes in the trabecular meshwork.

Late Congenital Glaucoma

The mesodermal remnants extend to the scleral spur (gonioscopically absent from the ciliary band) (Fig. 26.5).

As revealed by light microscopy, the anatomopathologic section appears as a loose fibrillated mesh for the most part comprising reticulin fibers and a few collagen fibers. There are also endothelial cells and sometimes very fine pigment granules. It extends over as a mesh through the chamber angle from the iris up to the Schwalbe line. The characteristic of these diffuse pathologic remnants is that they are positive for argentic coloration (Gomori and Del Río Ortega staining) methods.

Reticulin fibers are very thin, with neither periodical striation nor determined arrangement. They evolve to become collagen fibers. The genesis of these fibers may be so altered that they may be present in abnormally smaller or larger numbers.

The most adequate expression for this pathology is the persistence of a marked reticulogenesis in cases of congenital glaucoma, which may be morphologically evidenced by the richness of these fibers in both ligamentous and diffuse remnants.

Table 26.3 is an outline of normal and pathologic mesodermal remnants.

The structures described above were differentiated according to two criteria:

a.Topographic: (1) gonioscopy; (2) analysis and photography of the trabeculectomy specimen under surgical microscopy and slit-lamp; (3) optical surface microscopy (Zarate’s method), and (4) appraisal of the position of the elements of the chamber angle in magnified microscopy. Optical surface microscopy (Zarate’s method) (Fig. 26.6).

1.The tissue specimen is immersed in different alcohol concentrations (50°, 96°, and 100°) for dehydration.

2.Then this material is immersed in a bath of xylene where it becomes highly transparent.

3.At this time, vital staining is used (toluidine or thionine).

4.The specimen is reviewed with light microscopy and the image is documented.

b.Structural morphology is according to the staining properties of the different tissues.

– Hematoxylin-eosin can provide a topographic and morphologic criterion, but this technique is limited because it does not allow for differentiation between collagen and reticulin. This is the reason why other adequate additional techniques are used.

– PAS can be useful for neutral mucopolysaccharides, in the intertrabecular spaces.

– Masson’s stain is very useful to accurately determine the position and length of the ciliary muscle (red) and the spur with a topographic criterion. Masson’s stain is also useful for the differentiation of collagen structures, which acquire a blue coloration, as well as pathologic mesodermal remnants (diffuse or ligamentous), which stain in pink.

– Gomori and Del Rio Ortega (reticulin method) use silver embedding exclusively for reticulin [2]. Diffuse mesodermal remnants are stained in black. With this technique, given the large quantity of reticulin fibers, the reticular fibers surrounding the fibers of the ciliary muscle are also stained (see Chap. 15, Fig. 15.34a–d).